Induction of Inducible c AMP Early Repressor Expression

Induction of Inducible c. AMP Early Repressor Expression in Nucleus Accumbens by Stress or Amphetamine Increases Behavioral Responses to Emotional Stimuli

Introduction n c. AMP response element (CRE) is sensitive in many brain areas to drugs, stress, and reward increased CRE-mediated transcription in NAc (by drugs or stress) decreases behavioral response to +/- stimuli Blocking CRE transcription via CREB (CRE-binding protein) generally does the opposite

ICER n n n Inducible c. AMP early repressor (ICER) protein represses CREmediated transcription ICER = alternative to CREM (CREmodulator gene) ICER opposes CREB in behavior regulation to aid in adapting to aversive & rewarding stimuli

ICER n n n ICER = collective name for a group of proteins produced from the CREM/ICER gene 1 st described as the major CREM transcript in pineal gland during daynight flux of CREM expression Studies suggest ICER is involved in returning stimulated cells to the steady-state level

Why Amphetamines? n n n Amphetamines induce behavioral response by releasing DA from synapse DA release after AMPH administration is Ca 2+ independent Relies on cytosolic DA & vesicular DA permeating to neuronal cytoplasm from synaptic vesicles

Why Amphetamines? n n Repeated, intermittent Tx of rats w/ AMPH or cocaine enhances DA release from striatum & NAc Previously, AMPH induces CREmediated transcription w/in striatum

Materials & Methods

M&M n n n n Male Sprague Dawley rats Pair-housed 12 h light/dark cycle w/ dark @ 7 pm Procedures during the day Rats killed by rapid decapitation m. RNA isolated & purified m. RNA rev transcribed into c. DNA ICER quantified using real-time PCR

M&M n n n Amphetamine administered intraperitoneally @ 0. 5, 2, or 4 mg/kg 7 daily injections either saline or AMPH Acute = 6 days of saline + 1 day AMPH 7 d = 7 days of AMPH Acute stressed rats in conical sleeve for <60 min

M&M Unpredictable stressed rats: n Day 1: cage rotation 50 min @ 12 pm n Day 2: wet cage 60 min @ 11 pm, lights overnight n Day 3: lights off 180 min @ 12 pm, wet cage 15 min @ 3 pm n Day 4: cage rotation 50 min @ 7 pm, food/H 2 O deprivation overnight

M&M n n n Day 5: swim stress 3 min @ 1 pm, isolation housing overnight Day 6: restraint stress 60 min @ 11 am, lights off 120 min @ 3 pm Day 7: swim stress 4 min @ 10 am, restraint stress 60 min @ 12 pm

M&M n n n ICER m. RNA levels in NAc measured to acute stress Killed 4 h after stresses ICER & CREM c. DNAs amplified from rev transcribed m. RNA HSV (Herpes Simplex Virus) mediated CREM & ICER transcription was driven by a viral promoter Enhanced green fluorescent protein (GFP) used as control

M&M n n HSV vectors injected bilaterally in NAc Behavioral measures taken 48 -96 h after surgery 48 h after injection, rats given 4 mg/kg AMPH Brains harvested 3 h later & frozen on dry ice

M&M n n n GFP used as injection site marker & infected tissue micro dissected RNA purified, amplified, and rev transcribed CREB target = brain-derived neurotrophic factor (BDNF) measured using real-time PCR HSV-ICER vector was injected into rat NAc shell Expression verified using fluorescent immunohistochemistry

M&M n n PC 12 cells were infected w/ HSVLac. Z or HSV-ICER & protein harvested 12 h after ICER protein ID via Western Blot 90% HSV-Lac. Z or HSV-ICER & 10% HCR-GFP, 2 separate groups of rats injected into NAc Chromatin immunoprecipitation (Ch. IP) assays used to study ICER binding to promoter

M&M n n Locomotor activity measured in circular corridor via 4 photoelectric cells in every 90° of circle Spontaneous activity measured for 120 min in circular corridor chambers AMPH-stim rats given 2 - 60 min sessions 48 h after surgery before testing Test day: 60 min in corridor before ip injections of 2 or 4 mg/kg AMPH

M&M n n H 2 O removed w/ 1% sucrose in its place but removed 48 h before surgery Testing includes: • Isolation @ 5 pm w/ just food • @ 7 pm H 2 O & 1% sucrose bottles for 10 min • Neophobia: rats not pre-exposed & 30 min session

M&M n n Viral vectors injected into NAc Post op day 3: 24 h isolation Post op day 4: measured social behavior Cage mates together in 50 x 50 cm for 30 min • Watched for social grooming, exploring, playing, resting

M&M n n Force swim test rats in 25° H 2 O tubs for 15 min then 5 min Immobility: no struggling for 1 s (anti -depressive) Elevated plus maze for 5 min measured time in open arms Peanut butter chip neophobia: • Isolated rats 16 h before testing • PB chips in cup for 2 h in home cage during light phase

RESULTS

Figure 1

Results – Figure 1 AMPH on ICER overexpression n AMPH increased ICER m. RNA in striatum (3 h peak & 12 h normal) n AMPH regulation= dose dependent decreasing w/ repeated use • 1 st dose= 3 x increase • 7 th dose= 50% increase • Shown over 24 h

Figure 2

Results – Figure 2 Stress Tx n Increased ICER m. RNA 75% (1 -4 h peak after stress & 12 h normal) n Smaller compared to AMPH n Peak duration longer w/ acute restraint stress n Chronic restraint stress= no increase n 10 min forced swim or open arm confinement increased ICER m. RNA 4 h after stress

Results n Why HSV-ICER vector? • Expresses ICER under viral promoter control n n n Compare m. RNA levels of BDNF, CRE target gene, in NAc w/ HSV-ICER or HSV-Lac. Z 3 h after 4 mg/kg AMPH ICER decreased BDNF m. RNA Ch. IP experiment: CREM/ICER binding to BDNF promoter (ICER induced)

Results Further study: n n Injected HSV-ICER into NAc of CRELac. Z reporter mice 3 rd day (@ optimum HSV expression): 1 mg/kg AMPH • Boost Lac. Z expression n Observed 4 h later

Figure 3

Results – Figure 3 n n ICER caused decreased GFP CRE activity not affected by viral infection HSV-ICER inhibits CRE transcription in NAc No significance in CREM

Figure 4

Results – Figure 4 Locomotor activity n n n ICER overexpression in NAc: Decreased spontaneous activity during middle of test ICER enhanced AMPH-induced locomotor activity in entire test after 2 mg/kg dose 20 -60 min after AMPH= increase Higher dosage? Not significant

Figure 5

Results – Figure 5 Natural reward n n n ICER compared to Lac. Z expression ICER increased sucrose preference in familiarized animals ICER increased social reward or grooming time Depression-related behavior n n ICER created an anti-depressant effect Longer latency to immobility in forced swim

Figure 6

Results – Figure 6 Anxiety-related behavior n n n ICER increased anxiousness More time spent on closed arms of elevated plus maze Animals avoided unfamiliar tastes or neophobia (sucrose & PB chips) Rats frequently turned away from sucrose as a novel taste But…enhanced preference when familiar Neophobia is regulated by the NAc causing increased anxiety

Discussion n n Stress & AMPH increase ICER expression in striatum Mimic effect by viral-mediated gene transfer Gradual peaks in behavior response d/t delay in ICER induction so CREB can induce transcription in target genes - ICER translation ICER overexpression in NAc = increase response to AMPH

Discussion n n ICER affects other protein expression which affect neuronal function – cannot do it alone CREB-ICER competition @ CRE sites create behavioral response to stimuli ICER = negative feedback shutting down transcription normally induced by CREB Rats not as affected w/ repeated stimulation – CREB contributes to pathologic behavior d/t stress or chronic AMPH

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